Identification and molecular characterization of rice promoters conferring microspore preferred expression

Thesis for the Degree of Ph.D Identification and molecular characterization of rice promoters conferring microspore-preferred expression School of Applied Biosciences, Major in Agronomy The Graduate School Nguyen, Tien-Dung December 2015 The Graduate School Kyungpook National University Identification and molecular characterization of rice promoters conferring microsporepreferred expression Nguyen, Tien-Dung School of Applied Biosciences, Major in Agronomy The Graduate School Supervised by Professor Lee, Jeong-Dong Approved as a qualified thesis of Nguyen, Tien-Dung for the degree of Ph.D by the Evaluation Committee December 2015 Chairman Prof Song, Jong-Tae Prof Park, Soon-Ki Prof Jung, Ki-Hong Prof Park, Dong-Soo Prof Lee, Jeong-Dong The Graduate School Council, Kyungpook National University TABLE OF CONTENTS LIST OF TABLES II LIST OF FIGURES III LIST OF APPENDIX V ACKNOWLEDGEMENT VI CHAPTER GENERAL INTRODUCTION CHAPTER IDENTIFICATION AND CHARACTERIZATION OF MICROSPORE-PREFERRED GENES 19 INTRODUCTION 19 MATERIALS AND METHODS 20 RESULTS 27 DISCUSSION 56 CHAPTER FUNCTIONAL VERIFICATION OF MICROSPOREPREFERRED PROMOTERS ACTIVITY IN MICROSPORE 67 INTRODUCTION 67 MATERIALS AND METHODS 68 RESULTS 71 DISCUSSION 85 CHAPTER GENERAL DISCUSSION 87 REFERRENCES 93 I LIST OF TABLES Table Candidate genes exhibiting microspore-preferred expression 30 Table The most abundant CREs in RMP promoter region 34 Table Organ specific-CREs in RMP promoter region 35 Table Unique CREs in RMP promoter region 36 II LIST OF FIGURES Figure Diagram of rice (Oryza sativa L.) floral structure Figure Schematic diagram of the male gametophyte development in Arabidospsis Figure Female gametophyte development in Arabidospsis Figure Heat map analysis of expression patterns for rice microspore-preferred (RMP) genes 28 Figure Schematic diagram of destination vectors used for plant transformation 31 Figure Frequency of the most abundant CREs in RMP promoter region 39 Figure Frequency of organ specific-CREs in RMP promoter region 40 Figure Frequency of unique CREs in RMP promoter region 41 Figure Gus expression driven by RMP promoters during pollen development in rice 46 Figure 10 Confirmation T-DNA insertion in T2 rice transgenic plants 47 Figure 11 Gus expression driven by the RMP promoters in vegetative organs in rice 48 Figure 12 GUS expression driven by the RMP promoters during pollen developmental stages in Arabidopsis 53 Figure 13 GUS expression driven by the RMP promoters in Arabidospsis at seedling stage 54 Figure 14 The schematic diagram of prOsLSP10/RMP-SCP:dHA vectors used for genetic complementation analysis 73 Figure 15 The percentage of aberrant pollen grains from non-transformed scp-2 homozygotes (control) and transformed scp-2 hm harboring the proOsLPS10SCP:dHA 74 Figure 16 Complementation analysis of scp-2 homozygotes 75 III Figure 17 The percentage of aberrant pollen grains from non-transformed scp-2 homozygotes (control) and transformed scp-2 hm harboring the proRMP1SCP:dHA 79 Figure 18 The percentage of aberrant pollen grains from non-transformed scp-2 homozygotes (control) and transformed scp-2 hm harboring the proRMP2SCP:dHA 80 Figure 19 The percentage of aberrant pollen grains from non-transformed scp-2 homozygotes (control) and transformed scp-2 hm harboring the proRMP3SCP:dHA 81 Figure 20 The percentage of aberrant pollen grains from non-transformed scp-2 homozygotes (control) and transformed scp-2 hm harboring the proRMP6SCP:dHA 82 Figure 21 Silique production in complementing lines (transformed scp-2 hm) compared with scp-2 hm mutant background (control) and wild type plants 83 Figure 22 Confirmation of T-DNA insertion in the proRMP-SCP:dHA lines 84 IV LIST OF APPENDIX Appendix Primers used in this study 120 Appendix Medium composition used for rice transformation 122 Appendix Nucleotide sequences of the OsLPS10 gene 125 Appendix Nucleotide and protein sequences of the RMP1 (Os01g0533400) gene 129 Appendix Nucleotide and protein sequences of the RMP2 (Os01g0899100) gene 131 Appendix Nucleotide and protein sequences of the RMP3 (Os03g0381000) gene 133 Appendix Nucleotide and protein sequences of the RMP4 (Os04g0561900) gene 135 Appendix Nucleotide and protein sequences of the RMP5 (Os04g0650200) gene 137 Appendix Nucleotide and protein sequences of the RMP6 (Os06g0681100) gene 139 Appendix 10 Nucleotide and protein sequences of the RMP7 (Os07g0664600) gene 141 Appendix 11 Nucleotide and protein sequences of the RMP8 (Os12g0605900) gene 144 Appendix 12 Nucleotide and protein sequences of the RMP9 (Os12g0637100) gene 147 Appendix 13 Nucleotide and protein sequences of the RMP10 (Os12g0637900) gene 149 V ACKNOWLEDGEMENT First and foremost I would like to express my sincere gratitude to Prof Soon-Ki Park and Dr Sung-Aeong Oh for all the guidance, helpful advice during the whole period I would like to give a big thank to Prof Ki-Hong Jung in Kuyng Hee University for kindly provided microarray data profile, suggestions for my thesis I also would like to thank Prof Jong-Tae Song, chairman of my advisory committee, Prof Jeong-Dong Lee, and Prof Dong-Soo Park for their valuable suggestions and critical review of my thesis In addition, I would like to thank all members in Sexual Plant Reproduction Laboratory for their help Last, but not least, I wish to thank my wife, son, and family for their support, understanding and encouragement during all this time Nguyen Tien Dung VI Identification and characterization of microspore-preferred promoters in rice (Oryza sativa L.) Nguyen, Tien-Dung School of Applied Biosciences The Graduate School, Kyungpook National University Daegu, Korea (Supervised by Professor Lee, Jeong-Dong) (Abstract) Tissue-specific promoters are a very useful tool for manipulating gene expression in a target tissue or organ; however, their range of applications in other plant species has not been determined, to date In this study, I identified ten rice microspore-preferred (RMP1 to RMP10) promoters via meta-anatomical expression analysis I then investigated the expression of those promoters in transgenic rice (a homologous system) and Arabidopsis (a heterologous system) using GFP-GUS reporter genes As expected from microarray data analysis, all of the ten RMP promoters directed similar GUS expression pattern in anthers, GUS signals were detected from the microspore stage throughout the all stages of pollen development However, while four promoters, RMP2, RMP7, RMP9 and RMP10 did not direct GUS expressed in vegetative tissues such as leaf, stem, root at seedling stage, the other six promoters conferred GUS activity in those of seedlings These results suggest that RMP promoters could be expressed preferentially in microspore in rice In contrast, RMP promoters directed GUS gene showing distinct expression patterns in Arabidopsis In inflorescence, the RMP2, RMP3 and RMP8 promoters directed GUS expression in young buds but not in mature flowers GUS signals were observed only at uni-cellular and bi-cellular stages of pollen development On the other hand, promoters, RMP9 and RMP10, exhibited GUS expression in mature flowers at latepollen stages, tri-cellular and mature pollen Whereas, the other five promoters, including RMP1, RMP4, RMP5, RMP6, and RMP7 conferred GUS expression at all stages of pollen development, from uni-cellular throughout mature pollen The activity of these promoters was further examined in T2 seedlings As a result, seven promoters, except for RMP1, RMP2 and RMP10, showed GUS signals in shoot apical region or root tissues of seedlings In addition, analyzing promoter sequence revealed that the six most abundant CREs detected in RMP promoter regions such as ACGTATERD1, ARR1AT, CAATBOX1, GATABOX, MYBCORE, and DOFCOREZM Moreover, eleven CREs related to organs/tissues preferred expression Of them, anther or pollen specific CREs such as GTGANTG10, POLLEN1LELAT52, SITEIIATCYTC, 5659BOXLELAT5659 were identified Furthermore, to verify the activity of promoters in microspore I carried out a functional demonstration by performing a complementation analysis using a sidecar pollen (scp) mutant that displays developmental defects at the microspore stage Five promoters including the RMP1, RMP2, RMP3, RMP6 and OsLPS10 (rice late pollen specific promoter), which showed microspore expression in Arabidopsis, were also verified I found evidence that the OsLPS10, RMP1, RMP2, RMP3 and RMP6 promoters, which can be VII an applied promoter in Arabidopsis, are useful for directing gene expression in the early stages of pollen development The results indicate that those promoters can direct the expression of target genes during the stages of pollen development in rice, including early and late stages VIII RMP7 -2066 aaaattatcctttgagtctctctccatcttattcctgtattttttctgcagtccaatcaa -2006 actgccatttatatgtcttttctatgttttacaattctctgttttttacacattcttatc -1946 acaatcctgcattttctctataggcctgtttggttagtaccacattttgccacacttgtg -1886 gtatgttagtttgaccaaatgtggctagtgtttggtttgtttctataactttggcatgcc -1826 acactttctactcttttggtcccactagtcatagacccttttcttagccaaagtttgcca -1766 caagtgtggccatcaaattttgagccacacaatttgcatcttgccacacctttggtataa -1706 aaactgtggtaaattaaggtgggtatccaaacagtccatatgtcttcgtttaagtcattt -1646 tactagggttgttggtctgttccatccagagtggttggatgtgtacttcttgctatttca -1586 acaggtactacggctgtgtttagatccaAAGTTTGGATCCAAACTTcagtcctttttcat -1526 cacatcaacctgtcatacactcacaacttttcagtcacatcatctccaatttcaaccaaa -1466 atccaaacTTTGGATCCAActaaacacatccgtaacattaaaacttggcataaatacaaa -1406 tcatttcggatgcttttcttctcatcagacgcagagcctagaacagaacactgataatct -1346 gatactatcaatgagcatggctatatcaatgaatatgacgaAGAGTTCTATCgtcgatgc -1286 agtcttcaaatgcaagtttgatcagttatgcctatcgctttaatccccggttgtacttca -1226 tgacagAGAGTTCTATCctcgatccacactcttcagcttttaaaatgacaactttggtct -1166 ttgggtactaaaggggtccatcgattcttcctcaaaggtagatgaaatcgttctccgaca -1106 actatacatgcaagtgacatattataaacgaattgtatttctcgttgaaacgcacgggca -1046 cattactagtaagttataatttgtgaagcattttgagatgttttgtcatcttctgcgaag -986 ggcaatatgatatccaaaaggaagttctgagaaccttggactactattgtggtttttatt -926 tttctggacattattgtccaacagttggtcccctcatgtttaaaatgtggctccaccaac -866 gcacatgaaaggcgccgagtgctctcatcgcaggtagggccacaagcgctctcgtaaggg -806 cccgtgcatgtccaatgtggagcctaatgtccaagctgatctttctaagaccacttcatc -746 gcattgtgcatgctccaatggtacttctagattgataatatttgtcgttttagataaact -686 tgatgctaaacttaaaaatatttgactaatctctaatatctttttgtcttagaaatatgt -626 caaatgtataaattaatcttaaaaaatatttcaataaattcatatatttgttgacgtttc -566 tatacatattataattaaaaatattggttaaagttatttttttaagaccgtatccttatc -506 taaaacgataagaccaaccctcctgtacttcaaaaatttacgcaacacaaaacccactaa -446 ttattgtccttcatttggcccttcatatatgctctctccgtaaaaaaaaaaaaccttgtg -386 tccatgttcaaatttaggagtgggttttttttaagaacccgtaatataaaggattttgag -326 tttttgtttgcattttttgaccactcgtcttattcaaaaaatttgtgcaaatataaaaaa -266 tgaaaagttgtgcttaaagtattttggataataaagtaagtcaaaaaaataaataataat -206 ttcaaaattttttatgtaagacgagtggtcaaacagtgtaagaaaaaactcaaaatccct -146 tatattatgggatggagagagtaccattttcccccagttttttcaaacTATTTAActagc -86 ccctctctactgtgacctttggactcgagcttggaatcgaagaaccagtgaacacgagag -26 cacgacgccggtggcactcgttgaggATGATCAACGCAGCTGGGCAACTTCTTCTCAGgt +35 tgccatcgtcttcttcatattctctagagaaattatgatgattttgatctttttattttc +95 atacgtacacgcgttacagGGGGAGGAGCAGAGGTGTTCGTCCGATGTTCTCTTCCGGCC +155 TGGCCGATCGCTCCTTCTCCTCGTCGGCGTCAAGCTCCAGAAAGTTGGATGGGAAAGTGG +215 CCGTGATCACCGGCGCGGCGAGCGGCATCGGCGAGGCCACGGCGAAGGAGTTCGTCAGGA 140 +275 ACGGCGCCAAGGTCATCCTTGCCGATATCCAGGACGACCTCGGCCGCGCGGTGGCCGGCG +335 AGCTCGGCGCCGACGCCGCGTCGTACACGCACTGCGACGTCACGGTGGAGGCGGATGTCG +395 CCGCGGCCGTCGACCTCGCCGTGGCGCGCCACGGTCGTCTCGACGTCGTGTACAGCAACG +455 CCGGCATCGCCGGCGGCGCACCTCCGGCCACGCTCGCGGCGCTCGACCTCGACGACTACG +515 ACCGCGTCATGGCCGTCAACGCCAGGTCCATGGTGGCGTGCCTCAAGCACGCGGCGCGCG +575 TCATGGCGCCGCGCCGCGCCGGCTGCATCCTCTGCACGGCGAGCTCCACGGCGGTGCTCG +635 GCAACATCGGGCCCCTCGCGTACTCCATGTCGAAGGCGGCCGTCGTCGGCATGGTGCAGA +695 CGGTATTAGAAGTATAAtcttgttgttttgttttcagcatgtttatttgtatggtagtag +755 ctgagtagaagtcacatgtatgtgtgttgcatgccgtgagtgctagcttagtggttgtgc +815 tgcatggcttagtgagtagagagattggcagtgatcagtagtggctgatcgtgttgccag +875 gaggtgagagtacatctagctaattagttagccatgcatgcaaatgtggcgaagggatca +935 actcaggagcaggaagcggtcgaagtttgcatgggcgcatgcatcggagattgaccagga +995 gatattctgttagacgtgggtgagtttgttatctaggattcttgcatttctttaggctta +1055 agcaggtcatgtgcatctttaaatagagatcaatgtaatcagtttcagtgtgagaaaaaa +1115 acaaaaagaagtgctacgcacttgc Protein sequence MINAAGQLLLRGRSRGVRPMFSSGLADRSFSSSASSSRKLDGKVAVITGAASGIGEATAK 61 EFVRNGAKVILADIQDDLGRAVAGELGADAASYTHCDVTVEADVAAAVDLAVARHGRLDV 121 VYSNAGIAGGAPPATLAALDLDDYDRVMAVNARSMVACLKHAARVMAPRRAGCILCTASS 181 TAVLGNIGPLAYSMSKAAVVGMVQTVLEV Appendix 10 Nucleotide and protein sequences of the RMP7 (Os07g0664600) gene The promoter region that is infront of the start codon (ATG) and intron region were written in lowercases, uppercases indicate the exon regions The underlined sequences are primer sequences used for isolation of promoter region The putative cis-regulatory elements were highlighted as follows: DOFCOREZM (AAAG); POLLEN1LELAT52 (AGAAA); GTGANTG10 (GTGA); SITEIIATCYTC (TGGGCY, Y=C/T); ARR1AT (NGATT, N=A/C/T/G ); IBOXCORE (GATAA); WRKY71OS (TGAC); MYCCONSENSUSAT (CANNTG) 141 RMP8 -1422 tactgctgtcacatacagtgacaaaaacaattaaacctcatttgttcttccattgccctg -1362 aaagttggaataaccgtgaaaattggaataatcccagtaatgaacgaactaacctgacac -1302 tgacagtgaaaactaaagcaactgaggtcttgtttggcatagctccgggatcaAAGATTT -1242 TTTTGGagctgagtatttctagctacacaacaaatccatggatcatgataatatttggac -1182 gaattattttattcatatttttaataaaatataaaatttgcaagcactatattttaactt -1122 acaaacttcagatctaggatttgggatcttggagttgtgaatttgtgataaacataacct -1062 gaatttgtgtaaaacggggccgcaagtagtcttgttccagcccatgtgaaacagaccgta -1002 cataggggtcgtaataacaagtacaaccttttgacatacaaaatctaacggcgggttaaa -942 aatgagatgtgctaaaatttagtagattttttagggcttgttcactttgatgctattttt -882 aaccttaccaaatttgggtaaagttgccaaaaaaagtggctatatttagtttgctgccaa -822 attttggtactatataagaaattctgccaaTGCCAAAATTTTGGCAactatgccaaaatt -762 ttggtaatgccaaaatttggtAAGATTTTTTTGGcatcaaagtgaataggcccttagcta -702 ataaaaatacaattttgctatatATTTGTCGACAAATtttccaccaaaaatttgacagat -642 gtcgtAGTGTAATTACACTgtaactataatgtaacttgtacgtaactttcaaaaatatct -582 ccgtaacatgttatttcggtaaaatagaggttgtgggaacaaattctttcacatatacgt -522 gtgttgtgattgttttttcttcctcaccaaaataaatcctgtaatagatctaacgattca -462 aaattacatgaaacttacatataagttacactgtagttacatgCAAATTACAGTGTAATT -402 ACAtacaaattacAGTGTAATTACACTacaattgtactataattacatttgtcaaatttt -342 taggagaaaATTTGTCGACAAATatataggtggtcacataaaaaTATTTAAgagctgaga -282 tgggttgtaaaAGGGCCCATGGGCCCTggcccattaccaccacccatgcttgccgggctt -222 ccggcccacctacgtaagccccaccacgccgccaccgccaccaccgcaaccgtctccgct -162 tcctcctcctcctccccgtcgtgttcgcgcgcgacttccgtgacccacctccgccgcctc -102 tcgactcctccactctatatctcgagccctagaaaaaccctagctccagccactatctcc -62 actagcctacgtccaaggtcgtcttcgtcgtcgtctccgtctcgagcggccgggcgcgat -2 cgATGGCGTCGAGCTGCACCATGGTGCCGGCGGATTTGGTGGAGGAGAAGGCGGCGGCGG +69 CGGCGGTGGAGGAGGATGCGGGGAGTTACCTGCGCGCCGACCAGGTGGATCTCATGAGCC +129 TGGACTTCGAGATCGAGGAGAGGATGGCCGACAGGTTCCGCAAGCTCAACAGCGGCGGCG +189 TGGAGCGCGGCGACGAGGGGCCCAAGGCCGCGTGGGAGATCGACCTCTCCAAGCTGGAGA +249 TCGGCCACGTCGTCGAGCACGGCGACCACGGCACGCTCTTCCGCGGGAAGTACTACAGCC +309 AGGACGTCGCAGgtagaggaggatctcattctcatcgatcgatcgatcccatcctctttg +369 ttaaaccctaaacctctgcatgctgcatactaattgcatacggtgttttgggctgattga +429 gtcgatagaacctttgttatccatatatgcatgttctgatctggtagatgagattgtgtg +489 atgttgttttcttttaatagttcgttacaccatgctactatctttaattggaattgggat +549 cccatggccgtgttctttttagaacggttctgccatgaacattcttaaattcgacgggga +609 gttgtttcatgttgcattgatataggttggaagttggaagttggaaacctatatgtggat +669 aaaccaataatgtttttgtatggtggtctatgatcgacgtcgaacatccttgaattcaac +729 agaaaatcgcttcatgaaaacaattaactccaatggttaggttctcaaattttcgtcgtt 142 +789 gttgagaccctttgctatatatgaaagccgtatgaatggtagtgtcatgtatttcagttt +849 tggttatcaatcctaccacgttttatatatgtatttagattaaacgaatgagaaccatgt +909 gcatggatttgagttacttgcacagtattatataattttatcatgtttttacatatttat +969 taaaaaagaaattagtgatcataatcttatttggagatcttgacgatgttgaagatgaca +1029 tgttttattgctaccagaggaagtagctaatctacttctttctaatggtatgtgtagtga +1089 AACTGTTGGATTGGGGCGCAGAGGGCGATTCATCTGAAGACCAAATAGCTCATTTTAGAA +1149 CATCACTTAAGGAGGTGGTTGCTGTTTGGCATGAGTTCAACCATCCGAATATCACAAAGg +1209 tgatcatctctctgttcctagttgcagcaatccaatccatactgtgattttctttgcaat +1269 ggcattggcattgtcagctgtgcagaactaagatactgttatccattcgtgcacctgctc +1329 tttacatgtaattaggtgatattatatgccttctaagggatttcgttgttttgattcttt +1389 tgatccgttgcaaaattgttaaaatagtgctgctctgttttatttcttgaagtTCATCGG +1449 CGCATCGATGGGTACAACAAACCTTAATATTCCAAAAGATATTCCAGACCACAGTTCCAG +1509 GAAGGGTGCACGCACTGATCTGCCTGACAGAGCATGCTGTGTTGTGGTAGAATATCTCAC +1569 TGGGGGCACATTGAAACAGCATCTCATAAAGCATTACAGGAAGAACAAGAAGCTTCTATA +1629 TGAAGAGGTGGTTCGGCTTGCATTGGATTTGGCCAGAGGgtaattcagcaagttcgctat +1689 gcttctctctcttctgaatctgacgtttattaagcgttcgttccatcgtatctcttgttt +1749 cagattGAGCTTCCTGCACTCAAAAAAGATTGTGCATCGGGATGTCAAAAGTGAGAACAT +1809 GCTACTTGACCCACAGCTGAACCTTAAGATTGCTGATTTTGGTGTTGCTCGTCTCGTTGA +1869 GGCTCAGGATCCCAAGGATTTGACACGCACGACTGGAACACTTGGTTACATGGCCCCAGA +1929 Ggtaattatacacaactcacaacccagattttacagtatcctctcctgtattctgtgaat +1989 tcctttctccacgtcaaattcaaaaatttcttagataacgtgtcaacaccatgattattg +2049 catttaaaatgattctctcttaattatttgaaactccatcatacttcaggtgCTTGATGG +2109 AAAGCCATACAACAGAAAGTGCGATGTTTACAGTTTCGGCATCTGCCTATGGGAGACATA +2169 CTGCTGTGATATGCCCTATGGACCCTACTCAGACCTCAGCTTTGCAGACTTCTCATCGTT +2229 TGTTGTCCATAAGgtatcatcaagcaaacttgtctcacctcatttcgtcatatgatcatt +2289 tgcgttcttttggtcagactttgcgaagaaactctggatcatagtagtttctcctatacc +2349 ctgcattgcctcatatgatcatttgtgttcttttggtcagaatttgcgaagaatcaacaa +2409 aactactcttggatcacagtagtttctcctataccctgcatttcatcatatttgatcatt +2469 tatgtttgtgttgttttggacagaATTTGCGACCGGAGATCCCGGACTGCTGCCCGAGCG +2529 CAATGGCGAGCATCATGCGGAGATGCTGGGACGCCAACCCGGAGGTCCGGCCCGAGATGG +2589 AGGAGGTGGTGCGCCTCCTCGAGTCCCTGGACACGAGCAATGGCGGCGGCATGTTGCTGG +2649 AGAAGAAGAAGAAGAAGCATCCCGGCGGTGGATGCTTCTGCTTCTTCGTACCCCGCGCCG +2709 CGTAGgaaccatccattcacgtcaatgcttccatcttaatcagaatcatgtgctcctctt +2769 cctgtgtaaacttgattaggcagtttaggagtattattgttggatcctggcaccaacaaa +2829 aagcaagcgtcacgtcagtgtgtttagttgtctttgtttttttttcttcttccacgagat +2889 agatgagaattagattagctacggtttccttgctttgagatattagcattgaagcaagga +2949 ttggtaatttggatccatgaatggtgttgggatattggcattaaagcaaggattggtgag +3009 ttggatccatgaaaggtattggttgattgatt 143 Protein sequence MASRLLLLPRRRSRHGGASLLLARLLSSSSSEAGGGGAVEKVLVANRGEIACRVMRTARR 61 LGIPTVAVYSDADRGALHVRAADEAVRLGPPPARESYLNASAIVDAALRTGAKAIHPGYG 121 FLSESADFAQLCKAEGLTFIGPPPSAIRDMGDKSASKRIMGAAGVPLVPGYHGAEQDIEL 181 LKLEANKIGYPVLIKPTHGGGGKGMRIVQRPEDFVDSVLSAQREAAASFGINTLLVEKYI 241 TQPRHIEVQIFGDQHGNVIHLYERDCSLQRRHQKIIEEAPAPNVTAQFRSHIGEAAVSAA 301 KAVGYYSAGTVEFIVDTLSGEFYFMEMNTRLQVEHPVTEMIVGQDLVEWQIRIANGECLP 361 LSQEQVPLNGHAFEARIYAENVPRGFLPATGTLHHYRPVPSTATVRVETGVEEGDTVSMH 421 YDPMIAKLVVWGESRNAALVKLKNSLSNFQIAGLPTNVGFLQELAGHSAFEKGLVDTHFI 481 ERYQNDLLSTSTQALSGSHEAEELGAILAAACICKKDHVSSEVSLHDKKLSMWYAHPPFR 541 MHHFAKRLMEFELDRELGGSSDDLLKLSVTYRSDGTYFVETEDGSSPGLDVKVDSRGDHD 601 FRVDVGGLQTDVTLAFYSKDNCNHIHIWHGKHHHHYRQTLRAEQSPDDSSQPSASSEARS 661 HPKGSVLAPMAGLVVKVLLKDGARVEEGQPVMVMEAMKMEHVVKAPCAGYVEGLKATAGQ 721 QVFDSSVLFTVKENKPN Appendix 11 Nucleotide and protein sequences of the RMP8 (Os12g0605900) gene The promoter region that is infront of the start codon (ATG) and intron region were written in lowercases, uppercases indicate the exon regions The underlined sequences are primer sequences used for isolation of promoter region The putative cis-regulatory elements were highlighted as follows: DOFCOREZM (AAAG); POLLEN1LELAT52 (AGAAA); GTGANTG10 (GTGA); SITEIIATCYTC (TGGGCY, Y=C/T); ARR1AT (NGATT, N=A/C/T/G ); IBOXCORE (GATAA); WRKY71OS (TGAC); MYCCONSENSUSAT (CANNTG) 144 RMP9 -1450 ccttagctagtagtgagctactagtactagttgccatcgagaggtcatcttctccttatc -1390 tttctcttcttcttctcctctcaattaagatacatgcatcatgtgaaagcctcatgataa -1330 taccaggtcaagcagggcaacaactttatttctgttgattacaagttagtacaatgcgaa -1270 ctaaagaaaatttaggcattacttattcgagtcaaagcttgcccgtccacgacgccgata -1210 gccagtgagcttgacacgcgacctgccattgccactgccaaattttatggcattattcag -1150 aaaacttatactatatgctagttgattcagggaatgaaagtagctagactgctcctaatt -1090 cagagttcagaaagttaaGATGAATGGGTGGTcggttttcaaaaaaaaaaaaaGATGAAT -1030 GGGTGGTagcttattcagatttcacaaaagggaagagcaattgtggtattttaactcgga -970 tcaaccctctgaatgatgctgtcagttactggcactaacggttttacacagaaattaaac -910 aaaagtagaatctataaaggtgaatctgctgtctagtgcagcacaTAAAACGATATgagg -850 caatcattttgggggcaaatctgtgaacaagtggttaccaaatcgaccaaaaggtggcag -790 catgcttctctgtggcatgcttttggctagatcaagttgtgaaattgcgatctcctagct -730 agctttttcttcaaggccaccactgtgtttcaaagatgtagcgagtgatacatggatggg -670 caagtaacagtatgtgcattatactgaattctactagtaaaaaagtagagagctaagttt -610 cctttgttgaagaggtctgaatgatatgatatctttaacatatcttaacggaacaaatat -550 aaattgtgttatgcttaatatcatctggaatatactggcatatgataagcagttcacatt -490 acatcattTAAAACGATATccacgggtgatgtttcgacaaagaagaactgcaagcctgca -430 atgccagcacacatgtcgcaatcttttgtgaattggtggtgtgttttgtcagtcaaagta -370 ggcgcccatacaaatcagtagaaggcaaggcaaagcgatgtgagatttgtttgtccctcc -310 cttgcttggttggttgtacatgctgcgaatcgaagaataaatcagtagggatagaagagt -250 acttgtgttcaaagtcaacttgcaagtccttaatctccaagattgatccgcatctccaac -190 taaaccctctcctaatctaatcacggtctaatggcaccatctatggatattccccacaaa -130 ccattcagattagttcaagtcaacattaagatgcacgcaacactctccTATTTAAgagat -70 gtatcctttcttgcttgtccaacgaaacagagtagcacaagaagcagagtgcggctagct -10 cacagtcaccATGGGGATGCTGCGGTGGGGAGCTCACCTCCTCCTCCTCCTCCTCGCCGC +41 GGCGACGTGGACGTGCGCCGGCGCCGGCGCCGGGGTGACGAGCGAGTACCGGCGGAAGCT +101 GGAGGCGACGGTGGACATGCCGCTCGACGCCGACGTCTTCCGCGTGCCGCCGGGCTACAA +161 TGCTCCGCAGCAGgttcagtcttcttcagtctccacaaccacacaacgcgcgaccatctc +221 tcatctctttctttgtcatcaggtgCACATCACGCTGGGGGACCAGACGGGGACGGCGAT +281 GACGGTGTCGTGGGTGACGGCGAACGAGCTGGGGAGCAACACGGTCAGGTACGGGAGCTC +341 GCCGGAGAAGCTGGACCGCGCGGCGGAGGGGAGCCACACGAGGTACGACTACTTCAACTA +401 CACCTCCGGCTTCATCCACCACTGCACCCTCACCGGCCTCACCCACGCCACCAAGTACTA +461 CTACGCCATGGGCTTCGATCACACCGTCCGTACCTTCTCCTTCACCACGCCGCCCAAGCC +521 CGCTCCCGACGCCCCCTTCAAGTTCGGCCTCATCGgtgagtaacattacgccatgaattc +581 atcaccatttcgttcgtttcagttcatcatctcatcacatcaatttaaacaagtcattag +641 gagttgtgaacaaagtagatcgatcacatatgcaaacaaaaactggttcgataggattta +701 ggttaggtttagtctcacagtaatccgttgcattcaatgttgaatggtttcttgtcaagt 145 +761 ttttgcattaattatatatatcgggagactccaaagtcgctgcaaaccgccctcatccca +821 acgtcagtcagtgtcattactcaagtctgaatttcctctcgagtactcctacagtaccgt +881 actcaatcaagtaggtcagccgtgttgactatacatgcttcaaagtcatcaaaaaaacca +941 aaaatgtacgcagctggaattgatgaccatttgactgtgatgatcgtatggtatttgaaa +1001 ccaatgaagatgtagattacgttaacgcacataaaacaaaaggtcattattatataatta +1061 attaaattttaattattataaaattttcaaatggatatatttgacattttaatgcaattt +1121 ttatatagaaagttttgcatgactatttaatagttttaaatacatgctaacggaaaaaac +1181 tattaaacattttgtttcgtgtaaaaaatttctacatataggccgtgtttagttcacccc +1241 caagtccccaactactctaaactttcaactttccattacatcatatcacatccaaaactt +1301 tcctacactcataaacttttaattttttttcaggaactaaacacactcataaattgcttt +1361 aaaatatcaaatatgttcatttgacaaaatagtattagtaggagtactcccctacctcgc +1421 agtaaccgtttcagcgtgttactaatgtatgttagttgacttttttggaaaaaaaacaac +1481 tgtacgagttctgtcaaatgtagtacttagtacaaatacaaacgacgaaggacctgttta +1541 gttggcaaaaaaaaatctctacatgtcacatcagaacatcggatatacggacatatattt +1601 tattaaacatagtctaataataaaataaattatagattctgtcagaaaactgcgacatga +1661 atttatcaagcctaattaacatgtcattcgtaaatgtttactgtagcaccacattgtcaa +1721 atcatgacgcgattaggcttaaaagattcgtctcataatttcctgccgaactgtgatatt +1781 ggtttttttttatatttaatactctttatatgtgtccaaacatttaaaaaaaattgtttg +1841 gaactaaacaggccctaaattaatgaaatgaaatggatgagtgcacgcaggtgACCTCGG +1901 GCAGACGTTCGACTCGAACAGCACGCTGGCGCACTACGAGGCCAACGGCGGCGACGCCGT +1961 CCTCTTCGTCGGCGACCTCTCCTACGCCGACAACTACCCTCTCCACGACAACAACCGCTG +2021 GGACACGTGGGCGCGCTTCGTCGAGCGCAGCGTCGCGTACCAGCCATGGATCTGGACCGC +2081 CGGCAACCACGAGCTGGACTACGCGCCGGAGCTGGGCGAGACGGTCCCCTTCAAGCCCTT +2141 CACCCACCGGTACCCGACGCCGTACCGCGCCGCCGGCAGCACGGAGCCGTTCTGGTACTC +2201 CGTCAAGATCGCCTCCGCGCACGTCATCGTGCTGGCCTCCTACTCCGCCTACGGCAAGTA +2261 CACGCCGCAGTGGACATGGCTGCAGGAGGAGCTCGCCACCCGCGTCGACCGCAAGCTCAC +2321 CCCGTGGCTCATCGTGCTCATGCACTCGCCGTGGTACAACAGCAACAACTACCACTACAT +2361 GGAGGGGGAGACGATGCGCGTCCAGTTCGAGCGGTGGCTCGTCGACGCCAAGGTGGACGT +2421 GGTGCTCGCCGGCCACGTCCACTCGTACGAGCGCAGCCGGAGGTTCGCGAACATCGACTA +2481 CAACATCGTGAACGGCAAGGCGACGCCGGCGGCGAACGTGGACGCGCCGGTGTACATCAC +2541 GATCGGCGACGGGGGGAACATCGAGGGGATCGCCAACAACTTCACGGTGCCGCAGCCGGC +2601 CTACTCGGCGTTCCGGGAGGCCAGCTTCGGCCACGCCACGCTGGAGATCAAGAACCGGAC +2661 GCACGCGCACTACGCGTGGCACCGCAACCACGACGGCGCCAAGGCCGTCGCCGACGCCGT +2721 CTGGCTCACCAACCGCTACTGGATGCCCACCAACGACGATGTATAAttattaatcgactc +2781 caaccatctcaaacttcttaatttgtatcactgctatgctattgccattcttcttcttct +2841 ttagctttggataataaatactcctactatgcaataacaaatactactagcagagtctat +2901 cttgttaagccgtgagataattaatccccaaatgggacacttgccaacacatttggctac +2961 caaattggatataattaaaaatgtttatattttggagcaaagagagtagtacaatctaaa +3021 agggagtcacaaaaggattggtttctttggaatatttttcatgtggctttgtttgtaaca 146 Protein sequence MGMLRWGAHLLLLLLAAATWTCAGAGAGVTSEYRRKLEATVDMPLDADVFRVPPGYNAPQ 61 QVHITLGDQTGTAMTVSWVTANELGSNTVRYGSSPEKLDRAAEGSHTRYDYFNYTSGFIH 121 HCTLTGLTHATKYYYAMGFDHTVRTFSFTTPPKPAPDAPFKFGLIGDLGQTFDSNSTLAH 181 YEANGGDAVLFVGDLSYADNYPLHDNNRWDTWARFVERSVAYQPWIWTAGNHELDYAPEL 241 GETVPFKPFTHRYPTPYRAAGSTEPFWYSVKIASAHVIVLASYSAYGKYTPQWTWLQEEL 301 ATRVDRKLTPWLIVLMHSPWYNSNNYHYMEGETMRVQFERWLVDAKVDVVLAGHVHSYER 361 SRRFANIDYNIVNGKATPAANVDAPVYITIGDGGNIEGIANNFTVPQPAYSAFREASFGH 421 ATLEIKNRTHAHYAWHRNHDGAKAVADAVWLTNRYWMPTNDDV Appendix 12 Nucleotide and protein sequences of the RMP9 (Os12g0637100) gene The promoter region that is infront of the start codon (ATG) and intron region were written in lowercases, uppercases indicate the exon regions The underlined sequences are primer sequences used for isolation of promoter region The putative cis-regulatory elements were highlighted as follows: DOFCOREZM (AAAG); POLLEN1LELAT52 (AGAAA); GTGANTG10 (GTGA); SITEIIATCYTC (TGGGCY, Y=C/T); ARR1AT (NGATT, N=A/C/T/G ); IBOXCORE (GATAA); WRKY71OS (TGAC); MYCCONSENSUSAT (CANNTG) 147 RMP10 -2100 tctttaatccctaatatatgcagattaatggttgagatctgaaccctgtcaatgtgctac -2040 tagcatggtaatagtatagtagcttttcaagtaactaggtactcaaggatcaagatcaag -1980 catgcgAAATCTAATCTTTCAGAATCAGAGCAATGTTAaaatccAATCTTTCAGAAccag -1920 agcaatgtgaaatcgctcagatagaagctaataagaaaaaaaaaggaaaagaaaaatgtg -1860 cattgtcattgccattttgccatgacattatggtggagccacccagtacatgtcataccg -1800 tgtttagtaaagcgctaataaagcctttcaaaaggctggcctgaagtgacaaatgcttcc -1740 agacactggtatggtagcaaaacacaacacatgcatacttttacatgcatctatcagctg -1680 ctgatgctgcatgctactacagcactgcactgatcatcactagtggatggtagtagtgag -1620 agaacactgtaatggcatgaattttttcttctttttttatcaaaggaatggagggacaac -1560 gctctccatttctattagagaaaaaacaattttacgattcttgagaaggtatcatttttt -1500 ctattctaaatttaatacctattgatatctaggtactataagataacaaattttacatta -1440 aatttttgataccttctcaagtactgtaaaattacttttatagaaatggtaagcttgtac -1380 ggtacaaaagagtctttgcctgagacaagtccatggtagcacacacccttgaggctagtt -1320 atggtagagacgagagatggggacatggggtacatgtacagtaataatattgcacagaga -1260 gagagagagagagagagagagagagggcatgcatactccattttactcccacgagttaaa -1200 attgttggagtggtccacatgtcgatggcctttgctgtgttgacagcctggtaactggaa -1140 aaagggtagtacagtaagcaggcagagtaactgtagttcagattttgggacccacaatct -1080 gaattgtgaagcgaggcatgagacacgcgtgaaagaatgattggacggaccacatactaa -1020 ttgtcactagctagggtgtgttattcttcacttcttagtggagtactatgaaatactcat -960 aacttctatcgtcggtttattgtattaggagggatcacatccccttctagaatacatttt -900 ctttaggcggagggtttagaactttaaattagaaagcaattcatacaggcatgatgagTA -840 TTAAttccacataaaaaaatcaagggacagaattgatactactactaacttctcgttcaa -780 taaaagaacaacttttagaagcgattgtaggacgagtttgtataagcatttgtttagatt -720 tgtctgttaataaagttgatttttttttcgctaaagcacgattaaaagagctataaggaa -560 tcaggccaaccatgcactgacctgaccttggtaaagcactcttgcagttgcccttgctgt -500 cgtgccggaggcgtcgatagttggtcgccggcgtgatctaagcctaatagggttagagtc -440 aaactgagttgtgatgtgtttgtgttggactggttcgtcatgggctggacgtatgtgcgc -380 tagctttggtgggcctgcagccgaccaaggcccggcccactatgtacgttgtgtttcaac -320 tgcctggtactactagtatagataggtgtacatttactgcaaccgcgagatcagcggagt -260 tcgtttctcaatgtcgatcatctaatctcatcgatggagataagaagatgaaattcagcc -200 cttccttcaccgaaaagaaagaaaccaatcatataatatactgtatcttcgtcgttaatt -140 aattcgttatctccatatctctctctctatatatatactcgcagccctatgctctgcccg -80 cgcactgtaaacaaattactaatagattctccgacgatcgtagttgattgttcgtcaccg -20 agatccgacaaacgccatccATGGGTCGCGGCGGCCGCGGTGGAGgttagattcttcgat +41 cgaccttaattttgttcttcgatcgatcagtcgtgcattatttggtagatccggccgacc +101 gatcactgggtcaatatatcgctaataagtagtagcaacagcttaacttttgtgaattaa +161 attattagtataattaagctagccagtaactaaattgaattaattaattggtgcgtacgt +221 gcaGGAGGAGGAGGGAGGTCAAGCTTCCGCGGCAGCAGCACGGGCACGAAAGCAGTACCC 148 +281 CGTGCTGCTCCGGCGACCACGCCGGCGGCGAAGAGCACAGGCACAGCTCCGGCCACCAAG +341 AATACTTCCTCCGGCAGCAATGACTCCGTCATAGGATCCATCGGATCTGCTTTCTTTGAT +401 Ggtcagttagttattcaatttgcttactaaggctctattcactttgttgcaaaaaaaaca +461 ttatcaaattttggtattttagcatagttgccaaaattttagtaatttgtcaaaattttg +521 gcatgatttcttatatagttatcaaaatttggcagcaaattaaatgtagccatttttttt +581 gataactttatcaaaatttagtaaggttgaaaatgcatcaaagtgaacagaccctaaatt +641 tctagctagctccacaatgctatatactgctatatatacacttgctctccatgcatagat +701 gcaaattaatctactagccaaataccgtgctttaatttgctaccgattaaacaaagtaat +761 agtaattttttttagaataatttcatttatattgtataatactgatgaaatgtgatagaa +821 tcgtacatgtgcagagattattttataacattaaagtacattattaggtagttaatggaa +881 aatatgattgagtaggtgataaataaaaaaaatactatactaaatggagtatagatattt +941 gaccttagtttttttaatcagatttttgtcactggattatttgcttcgtgcttctgagaa +1001 atatgcatgatttatcaaaatacttttgtacctattgtattggtcgtacacctaccactt +1061 aattgcgaaatttattttccataatattaatttttcatgttaataagtaaataatcgagc +1121 cgctctcaaactggacggcataatcatgaagtgaaaatgcagGGTGGGGATGGGGCACGG +1181 GTTACGGGATGGTGCAGAGGGGGATGGACGCCGTCTTCGGCCCTCGCACCGTCAACGTCG +1241 TCGACGCCACGTCCACTTCGTCTTCTCCGGCGCCGGCGGCCGCCGCTGCGGCTCATCCAA +1301 TGTTGGATGCGTGCGGCGCCCACAAGAAGGCGTTCCAGGAATGCGTCGCCCAGCAAGGGA +1361 TCCACGTCAGCAGATGCCAGCCCTACCTCGACATGCTCAACGACTGCCGCCGGGACTCCG +1421 CCGCCTCCGCCGCCGTCGGCGTTGCCACCACCACCAGAATTCTCTGA Protein sequence MGRGGRGGGGGGRSSFRGSSTGTKAVPRAAPATTPAAKSTGTAPATKNTSSGSNDSVIGS 61 IGSAFFDGWGWGTGYGMVQRGMDAVFGPRTVNVVDATSTSSSPAPAAAAAAHPMLDACGA 121 HKKAFQECVAQQGIHVSRCQPYLDMLNDCRRDSAASAAVGVATTTRIL Appendix 13 Nucleotide and protein sequences of the RMP10 (Os12g0637900) gene The promoter region that is infront of the start codon (ATG) and intron region were written in lowercases, uppercases indicate the exon regions The underlined sequences are primer sequences used for isolation of promoter region The putative cis-regulatory elements were highlighted as follows: DOFCOREZM (AAAG); POLLEN1LELAT52 (AGAAA); GTGANTG10 (GTGA); SITEIIATCYTC (TGGGCY, Y=C/T); ARR1AT (NGATT, N=A/C/T/G ); IBOXCORE (GATAA ); WRKY71OS (TGAC); MYCCONSENSUSAT (CANNTG) 149 소포자 선호 프로모터의 탐색 및 분자적 특성규명 누엔티엔둥 경북대학교 대학원 응용생명과학부 (지도교수: 이정동) (초록) 조직 특이적 발현 프로모터는 식물의 조직 또는 기관에서 유전자 발현을 유도하기 위한 유 용한 도구이다 그러나 다른 식물 종에 활용을 위한 범위 혹은 한계는 현재까지 명확 치 않다 본 연구에서는 메타 유전체 발현 분석을 통해 rice microspore-preferred (RMP1 - RMP10) 프로모터 10종을 탐색으로 특성을 분석하였다 GFP-GUS 표식유전자를 이용하여 벼와 애기장대 (Arabidopsis) 식물의 형질전환을 수행하여, 벼 유래 프로모터들의 발현양상을 조사하였다 Microarray 분석에서 예측한 바와 같이, RMP 프로모터 10종 모두 약(anther) 유사한 GUS발현양상 을 보였다 GUS유전자는 화분 발달의 전 단계에 걸쳐 발현되었으며, 소포자 단계에서 최초 발현되 기 시작하였다 그러나 RMP2, RMP7, RMP9 및 RMP10 프로모터 4종의 조절을 받는 GUS유전자가 잎, 줄기, 뿌리 등 식물 조직에서 발현되지 않은 반면에, 나머지 프로모터 6종은 모든 조직에서 GUS 활성을 나타냈다 이러한 결과는 RMP 프로모터가 벼 소포자에서 우선적으로 표현됨을 보여 준다 반면에 RMP 프로모터는 애기장대에서 다른 GUS유전자 발현 양상을 보였다 RMP2, RMP3 와 RMP8 3종의 프로모터는 어린 봉오리에 GUS 표현을 보였으나, 성숙한 꽃에서는 발현되지 않았 다 GUS발현은 화분발달 단계 중 1세포성 단계 및 2세포성단계에서만 관찰되었다 다른 한편으로 는, RMP9와 RMP10 2종의 프로모터 성숙한 꽃의 3세포성 성숙 화분에서 GUS발현을 유도하였다 한편 RMP1, RMP4, RMP5, RMP6을 RMP7 5종의 프로모터는 1세포성 단계에서 성숙화분까지 모 든 화분발달단계에 걸쳐 GUS 발현을 유도하였다 이상의 10종의 프로모터들의 활성은 상기 T2 세 대에서 조사하였다 조사결과, RMP1, RMP2 및 RMP10 3종을 제외한 종 모두 발기인은 shoot의 분열조직, 말단 뿌리조직에서 GUS 발현을 보여 주었다 또한, RMP 프로모터는 6종의 cis-acting element (ACGTATERD1, ARR1AT, CAATBOX1, GATABOX, MYBCORE 및 DOFCOREZM)를 프로모터 영역에서 다수 함유하는 것으로 나타났다 또한, 이들 중 11종의 cis-acting element는 기관/조직 우세발현에 관련된 것으로 밝혀진 바 있다 특히 GTGANTG10, POLLEN1LELAT52, SITEIIATCYTC, 5659BOXLELAT5659들은 약/화분 특이적 특정 CRES로 동정된 바 있다 150 또한, 소포자 발달 단계에서 변이를 보이는 sidecar pollen (scp) 변이체를 이용하여, 벼 유래 프로모터의 화분발달 단계 특이적 발현향상을 활용한 돌연변이 회복여부 (genetic complementation analysis)를 조사하였다 Arabidopsis 에서 소포자 발현을 보이는 종의 프로모터 (RMP1, RMP2, RMP3, RMP6, OsLPS10)들은 애기장대의 sidecar pollen 변이를 정상으로 회복하는 것으로 확인되었다 본 연구의 결과는 이상의 종의 프로모터들이 쌍자엽식물 애기장대 화분발달의 초기 단계에서 유전자 발현을 특이적으로 조절한다는 강력한 증거이며, 주곡작물의 벼 화분발달의 초기 및 후기에서도 유용 외래유전자의 특이적 발현유도/조절에 활용할 수 있음을 보여주고 있다 151 PUBLICATIONS Published papers Nguyen TD, Oo MM, Moon S, Bae HK, Oh SA, Soh MS, Song JT, Kim JH, Jung KH, Park SK (2015) Expression analysis of two rice pollenspecific promoters using homologous and heterologous systems Plant Biotech Rep 9: 297-306 Oo MM, Bae HK, Nguyen TD, Moon S, Oh SA, Kim JH, Soh MS, Song JT, Jung KH, Park SK (2014) Evaluation of rice promoters conferring pollen-specific expression in a heterologous system, Arabidopsis Plant Reprod 27: 47-58 Nguyen TD, Oo MM, Soh MS, Park SK (2013) Bioengineering of male sterility in rice (Oryza sativa L.) Plant Breed Biotech 1:218-235 Bae HK, Oo MM, Jeon JE, Nguyen TD, Oh SA, Oh SD, Kweon SJ, Eun MY, Park SK (2013) Evaluation of gene flow from GM to non-GM Rice Plant Breed Biotech :162-170 Papers in preparation Nguyen TD, Moon S, Nguyen NTV, Goh Y, An G, Oh SA, Jung KH, and Park SK Genome-wide identification and analysis of rice genes preferentially expressed in pollen at early developmental stage Nguyen TD, Li B, Francisco A, Eo HJ, Oh SA, Song JT, Soh MS, Kim JH, Park SK T-DNA Tagged gametophytic mutants showing aberrant pollen phenotypes in Arabidopsis Nguyen TD, Moon S, Oo MM, Bae HK, Oh SA, Soh MS, Song JT, Kim JH, Jung KH, Park SK Identification and characterization of rice 152 microspore-preferred promoters that allows manipulation of gene expression at early stage of pollen development in Arabidopsis Nguyen TD, Oh SA, Park SK Pollen-mediated gene flow from GM to non-GM rice Nguyen TD, Li B, Jeon JE, Oh SA, Song JT, Kim JH, Park SK AtCOG gene is essential for pollen development in Arabidopsis Patents Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os8g0560700 gene and uses thereof Korean Patent No 10-1441916 Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os04g0638800 gene and uses thereof Korean Patent No 10-1441932 Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os11g0683800 gene and uses thereof Korean Patent No 10-1441933 Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os02g0741200 gene and uses thereof Korean Patent No 10-1479928 Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os01g0919200 gene and uses thereof Korean Patent No 10-1479946 153 Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os04g0317800 gene and uses thereof Korean Patent No 10-1441937 Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os04g0585900 gene and uses thereof Korean Patent No 10-1441945 Park SK, Oh SA, Oo MM, Jung KH, Tien DN Microspore or pollen specific expression promoter from Oryza sativa Os07g0247000 gene and uses thereof Korean Patent No 10-1462085 154 .. .Identification and molecular characterization of rice promoters conferring microsporepreferred expression Nguyen, Tien-Dung School of Applied Biosciences, Major... wife, son, and family for their support, understanding and encouragement during all this time Nguyen Tien Dung VI Identification and characterization of microspore-preferred promoters in rice (Oryza... construction, and identification of the microspore-preferred genes 20 Identification of promoter region and vector construction The sequences of selected genes were searched on the Rice Functional